P460: ERK1/2 INHIBITION OVERCOMES RESSITANCE TO BCL2 INHIBITION IN ACUTE MYELOID LEUKEMIA BY IMPACT ON MITOCHONDRIAL DYNAMICS AND FUNCTION
Authors: Priyanka Sharma, Sujan Piya, Natalia Baran, Anna Zal, Qi Zhang, Joanne Munck, Kim-Hien Dao, Martin Sims, Michael Andreeff, Gautam Borthakur
Journal: HemaSphere (2023)

Background: Activation of RAS/RAF/MEK/ERK (MAPK) pathway is common in myeloid malignancies and is a pervasive mechanism of inherent/acquired resistance to targeted inhibition of Bcl-2, mutated IDH and FLT3 in acute myeloid leukemia (AML) (Zhang et al., Sig Transduct Target Ther. 2022). ERK1/2 are terminal kinases in the MAPK pathway and may be suitable targets regardless of upstream mutational/non-mutational mechanisms of MAPK pathway activation. ASTX029 is a dual-mechanism inhibitor of ERK1/2 in clinical development. ASTX029 inhibits catalytic ERK1/2 activity and prevents feedback phosphorylation of MEK1/2, a potential mechanism of resistance. Oncogenic RAS drives ERK1/2-mediated phosphorylation of the dynamin-like mitochondrial fission GTPase Drp1, activating mitochondrial fission (Nagdas et al., Cell Rep. 2019). Aims: Therapeutic synergy of venetoclax (Bcl2i) and ASTX029 was evaluated in context of venetoclax resistance. As mitochondria-centered processes contribute to venetoclax resistance, we studied mitochondrial dynamics (fission/fusion) and function (ROS, metabolism) in the context of ERK1/2 inhibition and combined ERK1/2 and Bcl-2 inhibition. Methods: Synergy of Bcl2 and ERK1/2 inhibition was assessed using Annexin V assay followed by RNAseq analysis to identify molecular mechanisms. Results: Compound 27 (ERKi), a close analog of ASTX029 (Munck J et al., Mol Cancer Ther, 2021), was highly synergistic with Bcl2i [combination index (CI) of 0.008] in AML cells with intrinsic (OCI-AML3, RAS mut) and acquired (OCI-AML2 isogenic parental and Bcl2i resistant) resistance to Bcl2i. The observed synergy was also confirmed in primary samples (N=8) (bulk CD45+ and CD34+/38- leukemia initiating cells) obtained from patients with AML previously treated with Bcl2i combinations (CI:0.03-0.23). Mechanistically, the synergy was mediated by altered mitochondrial attributes including increased mitochondrial ROS, decreased Mcl1, and decreased membrane polarization. ERKi treatment resulted in decreased phosphorylation of Drp1(Ser616) and increased mitochondrial length. DRP1 colocalization with mitochondrial outer membrane TOM20 was decreased with ERKi treatment. Mitochondrial imaging with super-resolution microscopy confirmed that overexpression of phosphomimetic Drp1 (Drp1-Ser616Glu-Ser637Ala) resulted in shorter mitochondrial length, unlike phosphonull Drp1, which did not alter mitochondrial length (Fig.1). The apoptotic phenotype associated with synergy of Bcl2i and ERKi was partially reversed with overexpression of either Mcl1 or phosphomimetic Drp1. Transcriptome profiling revealed upregulation of 6 pathways in response to Bcl2i including oxidative-phosphorylation, electron transport chain, Myc targets, E2F targets, epithelial mesenchymal transition, and KRAS signaling. Interestingly, these pathways were downregulated after ERKi+Bcl2i treatment. Other mitochondria relevant transcripts downregulated with ERKi were OPA1, MFN1 (fusion), and MFF (recruits DRP1 to mitochondria). Finally, ERKi treatment alone and in combination with Bcl2i reduced basal oxygen consumption and ATP production in OCI-AML3 and OCI-AML2 Bcl2i resistant cells. In-vivo efficacy studies in NSG mice using RAS mutant AML PDX are ongoing. Summary/Conclusion: ERK1/2 inhibition demonstrates strong synergy with venetoclax and overcomes resistance to venetoclax. This synergy is largely mediated through effects on mitochondrial structural and functional dynamics. Our findings provide a strong rationale for clinical development of ERK 1/2 inhibitor ASTX029 in the treatment of AML and highlights the impact of targeting mitochondrial dynamics.Keywords: Acute myeloid leukemia, Mitochondria, Leukemia